Method for single-step attachment of a label to target molecules

ABSTRACT

Compounds and methods are provided for a single-step covalent attachment of a label to a molecule comprising forming a covalently attachable labeling reagent for alkylating the molecule. Then, combining the covalently attachable labeling reagent with a mixture containing the molecule, under conditions wherein the labeling reagent has reactivity with the molecule thereby forming a covalent bond.

[0001] This patent application is related to U.S. patent applicationSer. No. 09/767,794 filed on Jan. 23, 2001.

[0002] The described invention relates to compounds and methods forcovalently attaching a label to a target molecule. More specifically,the compounds are alkylating compounds having a reporter molecule andthe covalent attachment is performed by a one-step alkylation reaction.

BACKGROUND

[0003] The analysis and detection of minute quantities of substances inbiological and non-biological samples has become a routine practice inclinical and analytical laboratories. These detection techniques can bedivided into two major classes: (1) those based on ligand-receptorinteractions (e.g., immunoassay-based techniques) and (2) those based onnucleic acid hybridization (polynucleotide sequence-based techniques).

[0004] Immunoassay-based techniques are characterized by a sequence ofsteps comprising the non-covalent binding of an antibody and an antigencomplementary to it. Polynucleotide sequence-based detection techniqueshave been characterized by a sequence of steps comprising thenon-covalent binding of a labeled polynucleotide sequence or probe to acomplementary sequence of the analyte under conditions which permithybridization of the bases through Watson-Crick pairing, and thedetection of that hybridization.

[0005] The non-covalent binding of a labeled sequence or probe to acomplementary sequence of a nucleic acid is the primary recognitionevent of polynucleotide sequence-based detection techniques. Thisbinding event is brought about by a precise molecular alignment andinteraction of complementary nucleotides of the probe and target. It isenergetically favored by the release of non-covalent bonding freeenergy, e.g., hydrogen bonding, stacking free energy and the like.

[0006] In order to employ the non-covalent binding of a probe for thedetermination of a nucleic acid containing a target sequence, it isnecessary to be able to detect binding of the probe to the target. Thisdetection is effected through a signaling step or event. A signalingstep or event allows detection in some quantitative or qualitativemanner of the occurrence of the primary recognition event.

[0007] A wide variety of signaling events may be employed to detect theoccurrence of the primary recognition event. The signaling event chosendepends on the particular signal that characterizes the reportermolecule employed. Although the labeling reagent itself, without furthertreatment, may be detectable, more often, either the reporter moleculeis attached covalently, or bound non-covalently to a labeling reagent.

[0008] There are a wide variety of reporter molecules that may beemployed for covalent attachment to the labeling reagent ofpolynucleotide sequences useful as probes in nucleic acid detectionsystems. All that is required is that the reporter molecule provide asignal that may be detected by appropriate means and that has theability to attach covalently to the labeling reagent.

[0009] Reporter molecules may be radioactive or non-radioactive.Radioactive signaling moieties are characterized by one or moreradioisotopes of phosphorous, iodine, hydrogen, carbon, cobalt, nickel,and the like. Preferably the radioisotope emits .beta. or gamma.radiation, and has a long half-life. Detection of radioactive reportermolecules is typically accomplished by the stimulation of photonemission from crystalline detectors caused by the radiation, or by thefogging of a photographic emulsion.

[0010] Non-radioactive reporter molecules have the advantage that theiruse does not pose the hazards associated with exposure to radiation, andthat special disposal techniques after use are not required. Inaddition, they are generally more stable, and as a consequence, cheaperto use. Detection sensitivities of non-radioactive reporter moleculesmay be as high or higher than those of radioactive reporter molecules.

[0011] The ability to label DNA with a non-radioactive detectable markersimply and reliably makes it attractive for use in a wide variety ofmolecular and cellular biology applications. Some specific applicationsin which a non-radioactively labeled DNA or RNA probe can be usedinclude hybridization reaction procedures (southern, northern, slot, ordot blots, in situ hybridization), nucleic acid localization studies,DNA or RNA quantitation and DNase or RNase quantitation.

[0012] Both enzyme mediated and direct labeling protocols have beendeveloped to attach non-radioactive detectable tags such as thefluorescent compounds fluorescein and rhodamine (and others) to DNA.While these labeling methods have allowed non-radioactive detectionsystems to approach or even surpass the radioactive methods in terms ofsensitivity there remains significant disadvantages with each of thenon-radioactive labeling systems developed to date. 1) Enzymatic DNAlabeling systems require a number of reagents including both unlabeledand labeled nucleotide precursors, primers, and/or enzymes to facilitateDNA synthesis. Labeling efficiency is not easily controlled and for thetwo most common labeling reactions (nick translation and random priming)it is not possible to create a labeled probe that is the same size asthe starting DNA. 2) Direct labeling methods also have significantlimitations which include a lower efficiency of labeling resulting inreduced sensitivity, laborious multi-step labeling protocols, harshreaction conditions, variability from reaction to reaction, and unstablereactants.

[0013] Direct labeling methods have been developed for chemicallymodifying nucleic acids for use as detectable probes in hybridizationexperiments.

[0014] Sodium bisulfite may be used in the presence of a diamine tointroduce primary amines on cytosine residues which could then besubsequently modifed with a reporter group. Adarichev et al (Adarichev,V. A.; Vorobeva, N. V.; Grafodatskii, A. S.; Dymshits, G. M.; andSablina, O. V. (1995) Molecular Biology 29(3): 538-545) used4-aminohydroxybutylamine to transaminate cytosine residues in a similarfashion. DNA has been modified at the C-8 position of adenine or guanineusing a diazonium salt attached to biotin. The diazonium salt isgenerated in-situ with sodium nitrite then directly reacted with DNA. Inanother labeling procedure, the carcinogen 2-acetylaminofluorene wasmodified to the reactive compound N-acetoxy-2-acetylaminofluorene byLandegent et al and attached to the C-8 position of guanine. DNAmodified by this reagent was subsequently detected using antibodiesdirected against the modified guanosine. The reactive aldehyde at the C8position (N⁷-formyl group) of a ring-opened guanine has also been usedas a target for direct labeling using an aldehyde reactive nucleophilesuch as hydrazine attached to a detectable label.

[0015] These reagents have different limitations, some of theselimitations are multi-step synthesis, the ability to derivatize onlysingle stranded DNA, the need to use large amounts of reagent or otherharsh conditions to get adequate amounts of DNA modification, and themodification of amines involved in double-stranded DNA base pairing.

[0016] The techniques of Northern and Southern blotting are two of themost powerful and frequently used procedures in molecular biology. Yetthe necessary manipulations are time consuming and are not likely to beautomated under current technology. Often the polynucleotide (RNA, DNA)under analysis must first be fractionated by size, transferred onto asolid support and then treated through a series of steps to ensure onlyspecific binding of a probe. Detection of the hybridized productsusually depends on radiolabeling, heavy metal derivatization or antibodycomplexation. The methods of blotting have been a staple of basicresearch, and now also serve in an ever increasing number of commercialkits used to diagnose genetic, malignant, and infectious diseases.

[0017] In 1967 Belikova et al. (Belikova, A. M., Zarytova, V. F. andGrineva, N. I. (1967) Tetrahedron Letters, 37:3557-62) first describedmonoadduct alkylation of ribonucleosides and diribonucleoside phosphatesusing 2-chloroethylamine residues. While this work provided evidencethat ribonucleosides could be covalently modified with the alkylatingmustard derivative, the efficiency of the process was very low.Utilizing a multi-step process Frumgarts et al. (Frumgarts, L. A.;Kipriyanov, S. M.; Kalachikov, S. M.; Dudareva, N. A.; Dymshits, G. M.;Karpova, G. G.; and Salganik, R. I. (1986) Bioorg. Khim. 12(11):1508-1513) alkylated DNA using the nitrogen mustard4-(N-methylamino-N-2-chloroethyl) benzylamine, and subsequently attachedfluorescent labels to the amine that had been covalently attached to theDNA. This multi-step process required that the mustard and fluorescentlabel be used in a large molar excess to the DNA being labeled.

[0018] Quinicrine (acridine) is a DNA intercalating molecule which isalso fluorescent. Caspersson et al. used this molecule both with andwithout the attachment of a nitrogen mustard to obtain chemical andphysicochemical information about metaphase chromosomal structure. Inthis study, the fluorescent pattern obtained using quinicrine, whichcontains no alkylating group, produced a band pattern of the same typeas the quinicrine mustard. (Caspersson, T.; Zech, L.; Modest, E. J.;Foley, G. E.; Wagh, U.; and Simonsson, (1969) E. Experimental CellResearch 58 128-140).

SUMMARY

[0019] Utilizing the nucleic acid alkylating ability of the nitrogenmustards we have developed a series of labeling reagents consistingof 1) a nitrogen mustard moiety, 2) a cationic linker and 3) adetectable marker. A nucleic acid reactive nitrogen mustard derivativeused in the synthesis of these labeling agents can be the aromaticnitrogen mustard 4-[(2-chloroethyl)-methylamino]-benzaldehyde. Thisnitrogen mustard derivative was described in U.S. Pat. No. 2,141,090.

[0020] The ideal labeling method would combine one step simplicity withhigh efficiency labeling that results in a labeled product that remainsintact, stable and the same size as the starting DNA. To develop alabeling reagent with these characteristics we used nitrogen mustardshaving the ability to alkylate DNA in a one step reaction resulting inDNA containing a covalently attached adduct.

[0021] The procedure for labeling results in the formation of a chemicalbond between the compound and the polynucleic acid and/or protein. Thelabeling procedure consists of incubating the polynucleic acid with thesaid compounds in aqueous or non-aqueous solutions, followed byseparation of the labeled polynucleic acid from the unreacted labelingreagent. The extent of labeling can be controlled by regulating therelative amounts of labeling reagent and polynucleic acid, by adjustingthe length of the incubation, by controlling the temperature of theincubation, by controlling the absolute concentrations of polynucleicacid and labeling reagent, and by controlling the composition of theaqueous or organic solution using solvent, pH, ionic strength, andbuffers.

[0022] The labeled polynucleic acid can be used for several purposes:

[0023] 1) techniques to detect specific sequences of polynucleic acidsthat rely upon hybridization or binding affinity of the labeledpolynucleic acid to target nucleic acid or protein; including dot blots,slot blots, Southern blots, Northern blots, Southwestern blot, FISH(fluorescent in situ hybridization), in situ hybridization of RNA andDNA sequences, and newly developing combinatorial techniques in whichthe polynucleic acid is on a “chip” or multiwell or multislot device.

[0024] 2) labeling polynucleic acids that are delivered to cells invitro or in vivo so as to determine their sub-cellular and tissuelocation

[0025] 3) labeling oligonucleotides that are used as primer inamplification techniques such as PCR (polymerase chain reaction)

[0026] 4) quantitating polynucleic acids

[0027] 5) quantitating nucleases (including RNases and DNases) byfluorescence polarization or fluorescence dequenching.

[0028] 6) sequencing polynucleic acids

[0029] 7) directly detecting mutations

[0030] 8) covalently attaching reactive groups for use in anti-senseapplications

[0031] Also described is a method of functionalizing digoxin. Digoxin isfunctionalized by first oxidizing the diol functionality on the terminalsugar residue forming digoxin di-aldehyde, and subsequent reductivealkylation using sodium cyanoborohydride and a linking compound. Thelinker consists of an organic molecule with a primary amine and aprotected primary amine, which after removal of the protecting groupresults in a digoxin derivative with a primary amino group. The primaryamine can be transformed into a thiol reactive group such as ahaloacetamide, a maleimide, a disulfide, or a pyridyldithio; an aldehydeor ketone reactive group such as a thiosemicarbazide or a hydrazide; anamine reactive group such as a succinimidyl ester, isothiocyanate, orsulfonyl chloride; or a carboxylic acid reactive group such as adiazomethane or a haloacetyl. This methodology results in a digoxinmolecule that can be used to label proteins, peptides, polysaccharides,lipids, and other molecules of interest. The straight-forward synthesisfrom inexpensive precursors also lends utility to this unique method ofsupplying a digoxin label.

[0032] A method is provided for a single-step covalent attachment of alabel to a nucleic acid comprising forming a covalently attachablelabeling reagent for alkylating the nucleic acid. Then, combining thecovalently attachable labeling reagent with a mixture containing thenucleic acid, under conditions wherein the labeling reagent hasreactivity with the nucleic acid thereby forming a covalent bond.

[0033] A compound is provided that is a labeling molecule comprising acovalently attachable labeling reagent for alkylating a chemical in asingle-step reaction, thereby providing the chemical with a detectablereporter molecule.

[0034] In a preferred embodiment, a compound is provided having thestructure comprising

D-B-A

[0035] wherein, D is selected from the group consisting offluorescence-emitting compounds, radioactive compounds, haptens,immunogenic molecules, chemiluminescence-emitting compounds, andproteins; B is selected from the group of molecules having an affinityfor nucleic acids by interactions consisting of electrostatic, minorgroove binding, major groove binding, and intercalation; and, A isselected from the group of alkylating agents consisting of mustards andthree-membered ring derivatives.

[0036] In a preferred embodiment, a compound having the generalstructure comprising

[0037] wherein, D is selected from the group consisting offluorescence-emitting compounds, radioactive compounds, haptens,immunogenic molecules, chemiluminescence-emitting compounds, andproteins; R is selected from the group of alkyls and hydrogen; R′ isselected from the group of alkyls and hydrogen; n is an integer from 1to 20; m is an integer from 1 to 20; x is an integer from 1 to 5; and, Ais selected from the group of alkylating agents consisting of mustardsand three-membered ring derivatives.

[0038] Also provided is a kit comprising a receptacle containing acovalently attachable labeling reagent for alkylating a chemical in asingle-step reaction. Instructions for use are also provided with thekit. By the term instructions for use, it is meant a tangible expressiondescribing the reagent concentration for at least one assay method,parameters such as the relative amount of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

[0039] Reference is now made in detail to the preferred embodiments ofthe invention, examples of which are illustrated in the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040]FIG. 1(a) is an illustration of the chemical structure for3-bromo-1-(trifluoroacetamidyl)propane.

[0041]FIG. 1(b) is an illustration of the chemical structure forN,N-dimethyl-N-[N′-(tert-butoxycarbonyl)-3-aminopropylamine].

[0042]FIG. 1(c) is an illustration of the chemical structure forN-[N′-(tert-butoxycarbonyl)-3-aminopropyl]-N,N-dimethyl-3-aminopropylammoniumsalt

[0043]FIG. 1(d) is an illustration of the chemical structure forN-[N′-(5-(and-6)-carboxy-X-rhodaminecarbonyl)-3-aminopropyl]-N,N-dimethyl-3-aminopropylammoniumsalt.

[0044]FIG. 1(e) is an illustration of the chemical structure forN-[N′-(5-(and-6)-carboxy-X-rhodaminecarbonyl)-3-aminopropyl]-N,N-dimethyl-3-N″-{4-[(2-chloroethyl)-methylamino]-benzylamine}aminopropylammoniumsalt

[0045]FIG. 1(f) is an illustration of the chemical structure for digoxindialdehyde.

[0046]FIG. 1(g) is an illustration of the chemical structure for digoxinlabeling reagent.

[0047]FIG. 1(h) is an illustration of the chemical structureN-[N′-{4-[(2-chloroethyl)-methylamino]-benzylamine}-3-aminopropyl]-N,N-dimethyl-3-aminopropylammoniumsalt.

[0048]FIG. 1(i) is an illustration of the chemical structure forN-[N′-(5-(and-6)-carboxyfluoresceincarbonyl)-3-aminopropyl]-N,N-dimethyl-3-N″-{4-[(2-chloroethyl)-methylamino]-benzylamine}aminopropylammoniumsalt.

[0049]FIG. 1(j) is an illustration of the chemical structure for:N-[N′-(biotincarbonyl)-3-aminopropyl]-N,N-dimethyl-3-N″-{4-[(2-chloroethyl)-methylamino]-benzylamine}aminopropylammonium salt.

[0050]FIG. 1(k) is an illustration of the chemical structure forcarboxy-X-rhodaminecarbonyl/CPI2 labeling reagent.

[0051]FIG. 1(l) is an illustration of the chemical structure for CPI2moeity.

DETAILED DESCRIPTION

[0052] Definitions:

[0053] alkylating—a chemical reaction that results in the attachment ofan alkyl group to the substance of interest, a nucleic acid in apreferred embodiment.

[0054] alkyl group—an alkyl group possesses an sp³ hybridized carbonatom at the point of attachment to a molecule of interest.

[0055] anti-sense—oligonucleotide that has sequence complementary tospecific sequence of mRNA.

[0056] aziridine—A three-membered ring containing one nitrogen atom.

[0057] enzyme—enzymes are proteins evolved by the cells of livingorganisms for the specific function of catalyzing chemical reactions.

[0058] aqueous or non-aqueous solutions—Aqueous solutions contain water.Non-aqueous solutions are made up of organic solvents

[0059] bifunctional—A molecule with two reactive ends. The reactive endscan be identical as in a homobifunctional molecule, or different as in aheterobifucnctional molecule.

[0060] buffers—Buffers are made from a weak acid or weak base and theirsalts. Buffer solutions resist changes in pH when additional acid orbase is added to the solution.

[0061] combinatorial techniques—Techniques used to prepare and to screenextremely large pools of polynucleic acid sequences in which thesequences are immobilized in known positions on “chips”, multiwelldevices, multislot devices, beads, or other devices capable ofsegregating the polynucleic acid sequences.

[0062] CPI moiety—Family of which alkylate double stranded DNA withoutcross-reactions with single stranded DNA, RNA, proteins or othernucleophile containing groups. The drug is a derivative of a naturallyoccurring antibiotic which binds double stranded DNA in the minorgroove. All compounds in the CPI family include the functionality:1,2,8,8a-tetrahydro-7-methylcyclopropa-[c]pyrrolo-[3,2-e]indol-4(5H)-one

[0063] crosslinking—The chemical attachment of two or more moleculeswith a bifunctional reagent.

[0064] cyclopropane—A three-membered ring made up of all carbon atoms.

[0065] electrostatic interactions—The non-covalent association of two ormore substances due to attractive forces between positive and negativecharges.

[0066] episulfide—A three-membered ring containing one sulfur atom.

[0067] FISH—in situ hybridization technique in which the probe islabeled with a fluorescent marker.

[0068] fluorescence dequenching—technique monitoring the reappearence offluorescent intensity when said fluorescence that had been previouslyquenched from having been in close proximity to another fluorescentmolecule is released from close proximity to the other fluorescentmolecule.

[0069] hapten—A small molecule that cannot alone elicit the productionof antibodies to it. However, when covalently attached to a largermolecule it can act as an antigenic determinant, and elicit antibodysynthesis.

[0070] hybridization—Highly specific hydrogen bonding system in whichguanine and cytosine form a base pair, and adenine and thymine (oruracil) form a base pair.

[0071] In Situ Hybridization—hybridization using the labeled polynucleicacid probe taking place at the site of the immobilized polynucleic acidtarget within the context of the cell.

[0072] intercalating group—A chemical group characterized by planararomatic ring structures of appropriate size and geometry capable ofinserting themselves between base pairs in double-stranded DNA.

[0073] labeling—attachment of a reporter molecule via a chemical bond toa compound of interest such as a polynucleic acid or protein.

[0074] labeling reagent—a compound containing a reporting molecule thatcan be covalently attached to a polynucleic acid or a protein

[0075] minor groove binding group—A chemical group with an affinity forthe minor groove of double stranded DNA through non-covalentinteractions.

[0076] major groove binding group—A chemical group with an affinity forthe major groove of double stranded DNA through non-covalentinteractions.

[0077] mustards—include nitrogen mustards and sulfur mustards. Mustardsare molecules consisting of a nucleophile and a leaving group separatedby an ethylene bridge. After internal attack of the nucleophile on thecarbon bearing the leaving group a strained three membered group isformed. This strained ring (in the case of nitrogen mustards anaziridine ring is formed) is very susceptible to nucleophilic attack.Thus allowing mustards to alkylate weak nucleophiles such as polynucleicacids. Mustards can have one of the ethylene bridged leaving groupsattached to the nucleophile, these molecules are sometimes referred toas half-mustards; or they can have two of the ethylene bridged leavinggroups attached to the nucleophile, these molecules can be referred toas bis-mustards. Examples:

[0078] nitrogen mustard—A molecule that contains a nitrogen atom and aleaving group separated by an ethylene bridge, i.e. R₂NCH₂CH₂X whereR=any chemical group, and X−a leaving group typically a halogen. Ingeneral:

R₂NCH₂CH₂X

[0079] R=any chemical group

[0080] N=nitrogen

[0081] X=a leaving group, typically a halogen

[0082] aromatic nitrogen mustard

RR′NCH₂CH₂X

[0083] R=any chemical group

[0084] N=nitrogen

[0085] X=a leaving group, typically a halogen

[0086] R′=an aromatic ring

[0087] R=any chemical group

[0088] bis nitrogen mustard

RN(CH₂CH₂X)₂

[0089] R=any chemical group

[0090] N=nitrogen

[0091] X=a leaving group, typically a halogen

[0092] sulfur mustard

RSCH₂CH₂X

[0093] R=any chemical group

[0094] S=sulfur

[0095] X=a leaving group, typically a halogen

[0096] aromatic sulfur mustard

RSCH₂CH₂X

[0097] R=an aromatic ring

[0098] S=sulfur

[0099] X=a leaving group, typically a halogen

[0100] bis sulfur mustard

S(CH₂CH₂X)₂

[0101] S=sulfur

[0102] X=a leaving group, typically a halogen

[0103] oligonucleotide—a polynucleic acid with 30 or lessbase-sugar-phosphate groups.

[0104] oxirane—A three-membered ring containing one oxygen atom, alsocalled an epoxide.

[0105] protein—a molecule made up of 2 or more amino acids. The aminoacids may be naturally occuring or synthetic.

[0106] polynucleic acids—refers to a string of at least twobase-sugar-phosphate combinations. Natural nucleic acids have a phophatebackbone, artificial nucleic acids may contain other types of backbones,but contain the same bases. Nucleotides are the monomeric units ofnucleic acid polymers. The term includes deoxyribonucleic acid (DNA) andribonucleic acid (RNA). RNA may be in the form of an tRNA (transferRNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messengerRNA), anti-sense RNA, and ribosymes. DNA may be in form plasmid DNA,viral DNA, linear DNA, or chromosomal DNA or derivatives of thesegroups. In addition these forms of DNA and RNA may be single, double,triple, or quadruple stranded. The term also includes PNAs (peptidenucleic acids), phosphothionates, and other variants of the phosphatebackbone of native polynucleic acids.

[0107] polynucleotide—a polynucleic acid with more than 30base-sugar-phosphate groups.

[0108] photochemical—Refers to a reaction that requires a light sourcefor initiation of reaction.

[0109] reporter or marker molecules—Chemical (organic or inorganic)molecules or groups capable of being detected, and in some cases,quantitated in the laboratory. Reporter molecules include, but are notlimited to fluorescence-emitting molecules (which include fluoresceins,rhodamines, pyrenes, lucifer yellow, BODIPY®, malachite green,coumarins, dansyl derivatives, mansyl derivatives, dabsyl drivatives,NBD flouride, stillbenes, anthrocenes, acridines, rosamines, TNSchloride, ATTO-TAG®, Lissamine® derivatives, eosins, naphthalenederivatives, ethidium bromide derivatives, thiazole orange derivatives,ethenoadenosines, CyDyes®, aconitine, Oregon Green, Cascade Blue, andother fluorescent molecules), radioactive molecules, immunogenicmolecules, haptens, such as digoxin, affinity molecules such as biotinwhich binds to avidin and streptavidin, chemiluminescence-emittingmolecules, phosphorescent molecules, oligosaccharides which bind tolectins, or a protein or an enzyme such as alkaline phosphatase. Thereporter molecule may also contain radioactive atoms such as H³, C¹⁴,P³², P³³, S³⁵, I¹²⁵, I¹³¹, Tc⁹⁹, and other radioactive elements.

[0110] salts—Salts are ionic compounds that dissociate into cations andanions when dissolved in solution. Salts increase the ionic strength ofa solution, and consequently decrease interactions between polynucleicacids with other cations.

[0111] single-step covalent attachment—a reaction set up to take placeafter all of the reagents necessary to perform covalent attachment areplaced in contact with each other in a receptacle, without furthersteps.

[0112] target—The specific sequence of bases in a nucleic acid presentin an analyte whose presence is to be detected.

[0113] Dot Blots—Technique in which the polynucleic acid is immobilizedon a nylon membrane or nitrocellulose filter before being probed withlabeled polynucleic acid.

[0114] Slot Blots—Technique in which the polynucleic acid is immobilizedon a nylon membrane or nitrocellulose filter using a slot blot apparatusbefore being probed with labeled polynucleic acid.

[0115] Southern Blot—Technique in which the polynucleic acid (DNA) istransferred from an agarose gel to a nylon membrane or a nitrocellulosefilter before being probed with labeled polynucleic acid.

[0116] Northern Blot—Technique in which the polynucleic acid (RNA) istransferred from an agarose gel to a nylon membrane or a nitrocellulosefilter before being probed with labeled polynucleic acid.

[0117] Southwestern Blot—Technique in which protein is transferred froman acrylamide gel to a nylon membrane or a nitrocellulose filter beforebeing probed with labeled polynucleic acid.

[0118] R-chloride—Is the aromatic nitrogen mustard4-[(2-chloroethyl)-methylamino]-benzylamine

[0119] R-aldehyde—Is the aromatic nitrogen mustard4-[(2-chloroethyl)-methylamino]-benzaldehyde

[0120] One can determine whether or not a particular compound issuitable for the present invention by comparing the candidate compoundwith successful compounds illustrated in the examples. A suitablealkylating compound will alkylate a target molecule in a one-stepreaction. The examples demonstrate suitable methods and preparation ofcompounds for successful alkylation of target molecules. If a candidatecompound performs to at least 80% of the efficiency of the compoundsdescribed in the examples, the compound is a suitable one-stepalkylating compound.

[0121] According to a preferred embodiment of present invention, anucleic acid molecule with a nucleotide sequence, which may becomplementary to that of a target sequence, is converted into a labelingcompound by covalently attaching a reporter molecule. Several compoundsare described that are useful for attaching reporter molecules tonucleic acids and proteins. In a preferred embodiment a compoundsuitable for use with the present invention consists of one or more ofthe following three parts:

[0122] A—alkylating group—chemical functionalities that areelectrophilic, allowing them to become covalently attached to compoundsbearing a nucleophilic group. Alkylating reagents include mustards(nitrogen mustards and sulfur mustards ); and three-membered rings(aziridines, oxiranes, cyclopranes, activated cyclopropanes, andepisulfides)

[0123] B—Spacer—a connection between the alkylating group and thereporter molecule—a spacer group that includes alkanes, alkenes, esters,ethers, glycerol, amide, saccharides, polysaccharides, heteroatoms suchas oxygen, sulfur, or nitrogen, and/or a molecule that is cleavableunder physiologic conditions such as a disulfide bridge or anenzyme-sensitive group. The spacer may bear a net positive charge, or beany of the following: minor groove binders, major groove binders,intercalating groups, or other DNA-binding proteins or peptides such astranscription factors thereby providing the compound with an increasedaffinity for polynucleic acids. The spacer is provided to alleviatepossible molecular interference by separating the reporter molecule fromthe alkylating compound as well as nucleic acids after alkylation.

[0124] C—reporter molecule—a chemical moiety attached to the compoundfor purposes of detection.

[0125] The reporter molecule may be fluorescent, such as a rhodamine orflourescein derivative. The reporter molecule may be a hapten, such asdigoxin, or a molecule which binds to another molecule such as biotinwhich binds to avidin and streptavidin or oligosaccharides which bind tolectins. The reporter molecule may be a protein or an enzyme such asalkaline phosphatase. The reporter molecule may also contain radioactiveatoms such as H³, C¹⁴, P³², P³³, S³⁵, I¹²⁵, I¹³¹, Tc⁹⁹, and otherradioactive elements.

[0126] In another preferred embodiment are several compounds that areuseful for crosslinking polynucleic acids and proteins to othercompounds including reporter molecules, proteins, lipids, andpolynucleic acids. In a preferred embodiment a compound suitable for usewith the present invention consists of one or more of the followingthree parts:

[0127] A—alkylating group—chemical functionalities that areelectrophilic, allowing them to become covalently attached to compoundsbearing a nucleophilic group. Alkylating reagents include mustards(nitrogen mustards and sulfur mustards ); and three-membered rings(aziridines, oxiranes, cyclopranes, activated cyclopropanes, andepisulfides)

[0128] B—Spacer—a connection between the alkylating group and thereporter molecule—a spacer group that includes alkanes, alkenes, esters,ethers, glycerol, amide, saccharides, polysaccharides, heteroatoms suchas oxygen, sulfur, or nitrogen, and/or a molecule that is cleavableunder physiologic conditions such as a disulfide bridge or anenzyme-sensitive group. The spacer may bear a net positive charge, or beany of the following: minor groove binders, major groove binders,intercalating groups, or other DNA-binding proteins or peptides such astranscription factors thereby providing the compound with an increasedaffinity for polynucleic acids.

[0129] C—reactive group a chemical functionality capable of undergoingfurther chemical reactions. Reactive groups include, but are not limitedto alkylating groups, amines, alcohols, sulfhydryls, isothiocyanates,isocyanates, acyl azides, N-hydroxysuccinimides, sufonyl chlorides,aldehydes, epoxides, carbonates, imidoesters, carboxylates,alkylposphates, arylhalides (such as difluoro-dinitrobenzene),iodoacetamides, maleimides, aziridines, acryloyl chlorides,flourobenzes, disulfides, succinamides, carboxylic acids, and activatedcarboxylic groups.

[0130] In another preferred embodiment is a method for labelingpolynucleic acids using a compound suitable for use with the presentinvention consisting of one or more of the following three parts:

[0131] A—alkylating group—chemical functionalities that areelectrophilic, allowing them to become covalently attached to compoundsbearing a nucleophilic group. Alkylating reagents include mustards(nitrogen mustards and sulfur mustards); and three-membered rings(aziridines, oxiranes also known as epoxides, cyclopranes, activatedcyclopropanes, and episulfides)

[0132] B—Spacer—a connection between the alkylating group and thereporter molecule—a spacer group that includes alkanes, alkenes, esters,ethers, glycerol, amide, saccharides, polysaccharides, heteroatoms suchas oxygen, sulfur, or nitrogen, and/or a molecule that is cleavableunder physiologic conditions such as a disulfide bridge or anenzyme-sensitive group. The spacer may bear a net positive charge, or beany of the following: minor groove binders, major groove binders,intercalating groups, or other DNA-binding proteins or peptides such astranscription factors thereby providing the compound with an increasedaffinity for polynucleic acids.

[0133] C—reporter molecule—a reporter molecule—a chemical moietyattached to the compound for purposes of detection. The reportermolecule may be fluorescent, such as a rhodamine or flouresceinderivative. The reporter molecule may be a hapten, such as digoxin, or amolecule which binds to another molecule such as biotin which binds toavidin and streptavidin or oligosaccharides which bind to lectins. Thereporter molecule may be a protein or an enzyme such as alkalinephosphatase. The reporter molecule may also contain radioactive atomssuch as H³, C¹⁴, P³², P³³, S³⁵, I¹²⁵, I¹³¹, Tc⁹⁹, and other radioactiveelements.

[0134] Any of a large number of nucleic acid sequences may be employedin accord with this invention for use as probes in the detection oftarget molecules. Included, for example, are target sequences in bothRNA and DNA, as are the polynucleotide sequences that characterizevarious viral, viroid, fungal, parasitic or bacterial infections,genetic disorders or other sequences in target molecules that aredesirable to detect. Probes may be of synthetic, semi-synthetic ornatural origin. Probe molecules include both polyribonucleotides andpolydeoxyribonucleotides.

[0135] Although the presently preferred embodiment employs nucleic acidsof approximately 30 base pairs or longer, shorter nucleic acids may beused, provided they are capable of specifically and stably hybridizingto a target sequence. The nucleic acids may be designed to hybridize toeither the sense or antisense strand of a DNA duplex. When the target ismessenger RNA, however, the sequence of the probe should becomplementary to it.

[0136] Synthesis

[0137] The synthetic methodology used to prepare the labeling reagentsof the invention is described below. The structures of these compoundsare included in FIG. 1.

FIG. 1(a): Preparation of 3-bromo-1-(trifluoroacetamidyl)propane

[0138] To a solution of 3-bromopropylamine (2.19 g, 10.0 mmol, AldrichChemical Co., Milwaukee, Wis.) and triethyl amine (1.67 mL, 12.0 mmol,Aldrich Chemical Co.) in 60 mL methylene chloride at 0° C. in a 200 mLroundbottom flask equipped with a addition funnel was addedtrifluoroacetic anhydride (1.69 mL, 12.0 mmol, Aldrich Chemical Co.) in60 mL methylene chloride over a period of 20 minutes. The reaction wasstirred overnight, washed 1×10 mL 2% bicarbonate, 1×10 mL water, anddried over magnesium sulfate. Removal of solvent yielded 2.07 g (88.5%)product as amorphous crystals. H¹-NMR (CDCl₃)): ? 3.55 (m, 2H), 3.45 (m,2H), 2.17 (m, 2H).

FIG. 1(b): N,N-dimethyl-N-[N′-(tert-butoxycarbonyl)-3-aminopropylamine]

[0139] 3-dimethylaminopropylamine (251 μL, 204 mg, 2.00 mmol, AldrichChemical Co.) was combined with diisopropylamine (348 μL, 2.00 mmol,Aldrich Chemical Co.) in 2 mL tetrahydrofuran. BOC-ON (542 mg, 2.20mmol, Aldrich Chemical Co.) was added to the stirring reaction mixture.The reaction mixture was stirred at room temperature for 12 hours.Following removal of THF on a rotary evaporator the residue wasdissolved in 30 mL diethyl ether, washed 3×2 N NaOH, and dried overMgSO₄. Solvent removal yielded 359 mg (88.7%) product as a colorlessoil. H¹⁻-NMR (CDCl₃): δ 5.16 (bs, 1H), 3.76 (m, 2H), 2.30 (m, 2H), 2.21(s, 6H), 1.65 (m, 2H), 1.44 (s, 9H).

FIG. 1(c):N-[N′-(tert-butoxycarbonyl)-3-aminopropyl]-N,N-dimethyl-3-aminopropylammoniumcarbonate

[0140]FIG. 1(b) (344 mg, 1.70 mmol) and FIG. 1(a) ( 433 mg, 1.85 mmol)were combined in 250 μL anhydrous dimethylformamide (DMF), and incubatedat 55° C. for 48 hours. Product was precipitated from the reactionmixture by the addition of diethyl ether. Product was dried under vacuumyielding 686 mg (92.5%) product as a colorless oil. H¹-NMR (D₂O): δ 7.95(s, 1H), 3.45 (m, 2H), 3.35 (m, 4H), 3.20 (m, 2H), 3.10 (s, 6H), 2.10(m, 2H), 1.95 (m, 2H), 1.45 (s, 9H). The triflouroacetamide group wascleaved by dissolving the reaction product (179 mg, 0.409 mmol) in 1.0mL methanol and 0.5 mL water. Sodium carbonate (173 mg, 4.09 mmol) wasadded and the reaction was stirred at room temperature for 12 hours. Thecarbonate was removed by centrifugation. Product was dissolved inmethanol and precipitated by the addition of diethyl ether yielding 93.5mg (66.5%) product as a colorless solid. TLC: silica gel; water/aceticacid/ethyl acetate; 2/2/1; Rf=0.61, developed using Dragendorff'sReagent. H¹⁻NMR (CD₃OD): δ 3.37 (m, 4H), 3.15 (m, 8H), 2.73 (m, 2H),1.94 (m, 4H), 1.44 (s, 9H).

FIG. 1(d):N-[N′-(5-(and-6)-carboxy-X-rhodaminecarbonyl)-3-aminopropyl]-N,N-dimethyl-3-aminopropylammoniumdi-trifluoroacetate salt

[0141] To a solution of 5-(and-6)-carboxy-X-rhodamine, succinimidylester (125 mg, 198 μmol, Molecular Probes) in 0.5 mL DMF was added FIG.1(c) (63.6 mg, 198 μmol) and diisopropylethylamine (69.2 μL, 396 μmol,Aldrich Chemical Co.) in 3.0 mL DMF. The reaction was stirred at roomtemperature for approximately 1 hour. The product was isolated byprecipitation with diethyl ether. The tert-butoxy carbonyl (BOC) groupwas removed by dissolving the residue in triflouroacetic acid(TFA), andincubating at room temperature for 20 minutes. The TFA was removed undera stream of N₂, and the residue was purified via HPLC (C-18:acetonitrile/0.1% TFA) to yield 107 mg (63.7%) product as a red solid.TLC: silica gel; acetonitrile/10% acetic acid; 3/1; Rf=0.30. H¹-NMR(CD₃OD) ? 8.75 (m, 1H), 8.25 (m, 1H), 7.45 (m, 1H), 6.60 (s, 2H), 3.50(m, 8H), 3.15 (s, 6H), 3.05 (m, 4H), 2.65 (m, 4H), 2.20 (m, 4H), 2.10(m, 4H), 1.95 (m, 4H).

FIG. 1(e):N-[N′-(5-(and-6)-carboxy-X-rhodaminecarbonyl)-3-aminopropyl]-N,N-dimethyl-3-N″-{4-[(2-chloroethyl)-methylamino]-benzylamine}aminopropylammoniumtri-trifluoroacetate salt

[0142]FIG. 1(d) (39.5 mg, 0.0465 mmol) and4-[(2-chloroethyl)-methylamino]-benzldehyde (18.3 mg, 0.093 mmol, kindlyprovided by V.V. Vlassov, Institute of Bioorganic Chemistry, SiberianDivision of the Russian Academy of Sciences, Novosibirsk) were dissolvedin 550 μL methanol. To the stirring solution was added sodiumcyanoborohidride (2.9 mg, 0.0460 mmol, Aldrich Chemical Co.); thereaction was stirred at room temperature for 22 hr. Excess borohydridewas quenched with the addition of HCl (0.057 mmol). Solvent was removedon rotary evaporator, and the residue was purified via HPLC (C-18:acetonitrile/0.1% TFA) to yield 41.8 mg (87.3%) product as a red solid.TLC :silica gel; acetonitrile/10% acetic acid; 3/1; Rf=0.13. H¹-NMR(CD₃OD) ? 8.75 (d, 1H), 8.25 (dd, 1H), 7.45 (d, 1H), 7.35 (d, 2H), 6.80(d, 2H), 6.55 (s, 2H), 4.15 (s, 2H), 3.65 (m, 4H), 3.55 (m, 12H), 3.15(s, 6H), 3.10 (m, 2H), 3.00 (s, 3H), 2.65 (m, 2H), 2.15 (m, 8H), 1.90(m, 4H).

FIG. 1(f): Digoxin Dialdehyde

[0143] To a solution of digoxin (2.30 g, 2.95 mmol, Sigma Chemical Co.,St. Louis, Mo.) in 24 mL of chloroform:methanol (1:1) was added sodiumperiodate (0.699 g, 3.27 mmol, Aldrich Chemical Co.) and 1.0 mL water.The solution was stirred at room temperature for 18 hr. The sodiumiodate precipitate was removed via filtration. Following solvent removalon rotary evaporator the residue was dissolved in 50 mL chloroform,washed 1×8 mL water, and dried over magnesium sulfate. The solvent wasremoved on a rotary evaporator and the residue was recrystalized fromchloroform/hexane resulting in the formation of an oil. Crystalformation was initiated by the addition of a small amount of diethylether yielding 2.22 g (96.6%) amorphous, white solid. TLC: silica gel;acetonitrile/water; 1/1; Rf=0.88 after development with2,4-dinitrophenyl hydrazine (Aldrich Chemical Co.).

FIG. 1(g): Digoxin Labeling Reagent

[0144] To a solution of FIG. 1(f) (300 mg, 0.385 mmol) and sodiumcyanoborohydride (24.3 mg, 0.385 mmol, Aldrich Chemical Co.) in 35 mLmethanol in a 100 mL roundbottom flask equipped with an addition funnelwas added 4-[(2-chloroethyl)-methylamino]-benzylamine (27.0 mg, 0.099mmol, kindly provided by V.V. Vlassov, Institute of BioorganicChemistry, Siberian Division of the Russian Academy of Sciences,Novosibirsk) in 15 mL methanol at a rate of 10 drop/minute. The reactionwas stirred at room temperature for 18 hours. The reaction mixture wasconcentrated, dissolved in acetonitrile, and filtered to remove boronsalts. The residue was purified on a 20 g silica gel column. Excess FIG.1(f) was eluted in 100 mL acetonitrile and 100 mL 2% water inacetonitrile. Product was eluted in 200 mL 5% water in acetonitrile.Solvent removal on a rotary evaporator yielded 35.7 mg (38.1%). TLC:silica gel; acetonitrile water; 9/1; R_(f)=0.27, 0.35 corresponding tothe protonated and free forms of the aromatic nitrogen. H¹-NMR (CDCl₃)7.35 (d, 2H), 6.65 (d, 2H), 5.95 (s, 1H), 4.90 (m, 5H), 4.25 (m, 2H),4.05 (m, 2H), 3.80 (m, 2H), 3.70 (m, 2H), 3.35 (m, 2H), 3.20 (m, 2H),3.00 (s, 3H), 2.50-1.10 (m, 40 H), 0.95 (s, 3H), 0.80 (s, 3H).

FIG. 1(h):N-[N′-{4-[(2-chloroethyl)-methylamino]-benzylamine}-3-aminopropyl]-N,N-dimethyl-3-aminopropylammoniumtetra-trifluoroacetate salt

[0145]FIG. 1(c) (123 mg, 0.382 mmol) and4-[(2-chloroethyl)-methylamino]-benzaldehyde (75.5 mg, 0.382 mmol,kindly provided by V.V. Vlassov, Institute of Bioorganic Chemistry,Siberian Division of the Russian Academy of Sciences, Novosibirsk) weredissolved in 9 mL methanol. Sodium cyanoborohydride (24.0 mg, 0.381mmol, Aldrich Chemical Co.) was added. The reaction was stirred at roomtemperature for 18 hours. Solvent was removed from the reaction mixture,the residue was dissolved in TFA, and incubated for 20 minutes at roomtemperature to remove the BOC protecting group. The TFA was evaporatedunder a stream of nitrogen, and the residue was purified via HPLC (C-18:acetonitrile/0.1% TFA) to yield 85.0 (27.9%) as a yellow oil. TLC:silica gel; dimethylfonnamide/acetic acid/water; 1/2/2; Rf=0.31.

FIG. 1(i):N-[N′-(5-(and-6)-carboxyfluoresceincarbonyl)-3-aminopropyl]-N,N-dimethyl-3-N″-{4-[(2-chloroethyl)-methylamino]-benzylamine}aminopropylammoniumtri-trifluoroacetate salt

[0146]FIG. 1(h) (117 mg, 0.147 mmol) and 5-(and-6)-carboxyfluoresceinsuccinimidyl ester (69.8 mg, 0.147 mmol, Molecular Probes, Eugene,Oreg.) were dissolved in 2.0 mL DMF. Sodium carbonate (31.0 mg, 0.292mmol) was added, and the reaction was stirred at room temperature for 48hours. The product was isolated by precipitation with diethylether.Product was purified by recrystalization from 10% methylsulfoxide in DMFand diethyl ether. TLC: silica gel; acetonitrile: 10% acetic acid; 3/1;Rf=0.43.

[0147]FIG. 1(j):N-[N′-(biotincarbonyl)-3-aminopropyl]-N,N-dimethyl-3-N″-{4-[(2-chloroethyl)-methylamino]-benzylamine}aminopropylammoniumtri-trifluoroacetate salt

[0148]FIG. 1(h) (10 μg, 12.5) and NHS-biotin (4 μg, 12.5 mol, PierceChemical Co., Rockford, Ill.) were dissolved in mL DMF. Diisopropylethyl amine (2 μL, Aldrich Chemical Co.) was added and the reaction wasstirred at room temperature overnight. The product was isolated byprecipitation with diethyl ether, and purified via HPLC (C18: 0.1%TFA:acetonitrile). TLC silica gel: 3/1, acetonitrile/50 mmol ammoniumacetate pH4) Rf=0.135.

FIG. 1(k): carboxy-X-rhodaminecarbonyl/CPI2 labeling reagent

[0149]FIG. 1(d) (66.5 μg, 0.078 μmol) in 50 μL dimethylformamide wascombined with CPI2 (50.0 μg, 0.078 mol, Epic Pharmaceuticals, Seattle,Wash.) and 1 μL of diisopropylethylamine (2% solution in DMF, AldrichChemical Co.). The reaction was complete after 2 hours at roomtemperature, as determined by TLC: silica gel; acetontrile/50 mMammonium acetate pH 4.0; 3/1; Rf product=0.12, Rf FIG. 1(d)=0.0.

EXAMPLE 1 Labeling Reagents can be Covalently Attached to DoubleStranded DNA (dsDNA)

[0150] By means of the procedure set forth below covalent attachment ofthe labeling reagents to double stranded plasmid DNA (pDNA) isdemonstrated. Detectable non-radioactive labels (fluorescein, rhodamine,and digoxin) coupled to nitrogen mustards (see synthesis section) werecovalently attached to polynucleic acids. Unincorporated labelingreagent is removed from labeled DNA by ethanol precipitation in 0.5 MNaCl.

[0151] (1) Materials

[0152] 1. pDNA (3.3 μg/μL pCIluc; prepared according to Danko, I.,Williams, P., Herweijer, H. et al. Hum. Mol. Genetics (1997) in press.)

[0153] 2. FIG. 1(e)

[0154] 3. FIG. 1(i)

[0155] 4. Hepes buffer (100 mM, pH 7.5)

[0156] 5. NaCl (5M aqueous solution)

[0157] 6. Ethanol (100%, Pharmco, Brookfield, Conn.)

[0158] 7. 70% Ethanol

[0159] 8. Photo-documentation camera equipped with a photo-documentationhood (FB-PDC-34 and FB-PDH-1314, FisherBiotech, Pittsburgh, Pa.)

[0160] 9. Ethidium bromide (Acros Organics, Pittsburgh, Pa.)

[0161] (2) Reaction Protocol

[0162] 0. For negative control purposes 5.0 μg portions each of FIG.1(e) and FIG. 1(i) were incubated in 200 μL Hepes buffer for 48 hours tohydrolyze the nitrogen mustard. These samples are used as negativecontrols; they no longer contain an alkylating group.

[0163] 1. FIG. 1(e) (1.0 μg), FIG. 1(i) (5 μg), hydrolyzed FIG. 1(e)(1.0 μg), and hydrolyzed FIG. 1(i) (5.0 μg) were combined with plasmidDNA (5 μg) in 150 μL Hepes buffer. The reactions were vortexed to mix.

[0164] 2. The reactions were incubated at 37° C. for 1 hour.

[0165] 3. 15 μL 5M NaCl was added to each tube, the solutions wasvortexed to mix, and 2 volumes ethanol was added.

[0166] 4. The reactions were incubated at −10° C. for 10 minutes, andcentrifuged at 1200 rpm for 5 minutes. The supernatant was decanted, andthe pellets were washed with 70% ethanol.

[0167] 5. The pellets were dissolved in 100 μL Hepes buffer. 0.4 μglabeled DNA from each reaction was run on a 1% agarose gel.

[0168] 6. A photograph was taken On a UV lightbox prior to ethidiumbromide staining (3 second exposure), and after ethidium staining (1second exposure).

[0169] (3) Results

[0170] The covalent attachment of the labeling reagents is demonstratedby visualization of DNA bands without ethidium bromide staining. Thelabeled DNA in lanes 3 and 5 is visible due to the fluorescence of thecovalently attached rhodamine, and fluorescein respectively. Theunlabeled control plasmid in lane 2 is not visible, nor is the plasmidDNA incubated with hydrolyzed FIGS. 1(e) and (i) (lanes 4 and 6respectively). Thus when the mustard moiety on the labeling reagents ishydrolyzed no labeling of DNA is observed.

[0171] Now the ladder marker in lane 1 and the unlabeled plasmid in lane2 are visible; as well as the DNA incubated with hydrolyzed labelingreagents. The integrity of the labeled plasmids is demonstrated bycomparing the ratio of supercoiled to relaxed circular plasmid DNA inthe unlabeled controls with that of the labeled plasmids. Any nickingwill result in an increase in relaxed circular plasmid DNA. Analysis ofthe photograph reveals nicking levels to be low.

EXAMPLE 2

[0172] Labeling reagents can be covalently attached to single strandedDNA (ssDNA)

[0173] (1) Materials

[0174] 1. M13 bateriophage M13mp18 ssDNA (Panvera Corporation, Madison,Wis.)

[0175] (2) Procedure and Results

[0176] The ssDNA was labeled using FIG. 1(e) according to example 1using a 30 minute incubation period at 37° C., and a labeling reagent:DNA ratio of 0.2:1. The labeled ssDNA was analyzed by agarose gelelectrophoresis, 1.0 μg of DNA per lane.

[0177] (3) Results

[0178] Lane 3a contains labeled ssDNA, and lane 7a contains unlabeledssDNA before ethidium bromide staining. Lane 3b contains labeled ssDNA,and Lane 7b contains unlabeled ssDNA after ethidium bromide staining.Incorporation of the labeling compounds is demonstrated by the observedfluorescence of labeled ssDNA, and the absence of unlabeled ssDNA priorto ethidium staining. The integrity of the labeled DNA is demonstratedby comparing the ethidium stained unlabeled control ssDNA with theethidium stained labeled ssDNA.

EXAMPLE 3

[0179] Labeling Reagents can be covalently attached to RNA

[0180] (1) Materials

[0181] 1. calf liver tRNA (Boehringer Mannheim Corporation,Indianapolis, Ind.)

[0182] 2. 900b in vitro transcribed RNA (0.3 μg/mL)

[0183] 3. FIG. 1(e)

[0184] (2) Procedure

[0185] The RNAs were labeled using FIG. 1(e) according to example 1using a 30 minute incubation period at 37° C., and a labeling reagent:RNA ratio of 0.2:1. The labeled RNAs were analyzed by agarose gelelectrophoresis, 1.0 μg of RNA per lane.

[0186] (3) Results

[0187] Lane 1a contains labeled tRNA, lane 2a contains labeled in vitrotranscribed RNA, lane 5a contains unlabeled tRNA, and lane 6a containsunlabeled in vitro transcribed RNA prior to ethidium bromide staining.Lane 1b contains labeled tRNA, lane 2b contains labeled in vitrotranscribed RNA, lane 5b contains unlabeled tRNA, and lane 6b containsunlabeled after ethidium bromide staining. Covalent attachment of thelabeling reagent to the RNAs is evident because RNA incubated withreagent shows visible fluorescence without ethidium bromide staining.The labeled RNA bands and the unlabeled RNA bands both migrate to anequal extent in the agarose gel, thereby demonstrating that the covalentattachment of the labeling reagent does not harm the RNA.

EXAMPLE 4

[0188] Labeling reagents can be covalently attached to linear doublestranded DNA

[0189] (1) Materials

[0190] 1. λ DNA/hind III fragments (Life Technologies Inc.,Gaithersburg, Md.)

[0191] (2) Procedure

[0192] The linearized λ DNA was labeled using FIG. 1(e) according toexample 1 using a 30 minute incubation period at 37° C., and a labelingreagent: DNA ratio of 0.2:1. The labeled linearized dsDNA was analyzedby agarose gel electrophoresis, 1.0 μg of DNA per lane.

[0193] (3) Results

[0194] Lane 4a contains labeled linearized dsDNA, lane 8a containsunlabeled linearized dsDNA prior to ethidium bromide staining. Lane 4bcontains labeled linearized dsDNA, and lane 8b contains unlabeledlinearized dsDNA after ethidium bromide staining. Covalent attachment ofthe labeling reagent to the linearized dsDNAs is evident becauselinearized dsDNA incubated with reagent shows visible fluorescence priorto ethidium bromide staining. The labeled linearized dsDNA bands and theunlabeled linearized dsDNA bands both migrate to an equal extent in theagarose gel, thereby demonstrating that the covalent attachment of thelabeling reagent does not harm the linearized ds DNA.

EXAMPLE 5

[0195] CPI2 based labeling reagents can be covalently attached to DNA

[0196] (1) Materials

[0197] 1. FIG. 1(k)

[0198] 2. pDNA (pCIluc; prepared according to Danko, I., Williams, P.,Herweijer, H. et al. Hum. Mol. Genetics (1997) in press.)

[0199] (2) Procedure and Results

[0200] pDNA was labeled using FIG. 1(k) with a labeling reagent to DNAratio of 1:1. The procedure for DNA labeling given in example 1 wasfollowed, however the incubation period was increased to 12 hours. Thecovalent attachment of the CPI2-based reagent (FIG. 1(k)) wasdemonstrated by agarose gel electrophoresis. The DNA band was visibleprior to ethidium bromide staining.

EXAMPLE 6

[0201] Incorporation of labeling reagents onto the polynucleotide isproportional to the amount of labeling reagent used

[0202] The extent of labeling reagent incorporation into thepolynucleotide can be controlled by regulating the ratio of labelingreagent to polynucleotide.

[0203] (1) Experimental Protocol

[0204] pDNAs (pCIluc) were labeled according to the procedure in example1 using FIG. 1(e) as the labeling reagent. DNA was labeled at thefollowing weight to weight ratios (labeling reagent: DNA): (0.01:1),(0.02:1), (0.05:1), (0.10:1), (O.20:1), (O.50:1), and (1:1). 0.2 μg DNAsample were analyzed on an agarose gel. The remaining portion of labeledDNA was analyzed on a Beckman DU6 UV/visible spectrophotometer (BeckmanInstruments, Inc. Arlington Heights, Ill.).

[0205] (2) Results

[0206] Extent of incorporation was observed by visualization of anagarose gel prior to ethidium staining. Fluorescence was visible for alllabeling ratios. An increase in the intensity of the fluorescence fromthe lowest to highest labeling reagent to DNA ratios was also observed.The absolute extent of label incorporation was determined by measuringthe absorbency of the purified samples at 576 nm (λ_(max) for5-(and-6)-carboxy-X-rhodamine, Molecular Probes Inc.). The absorptionintensity at 576 nm was divided by the absorption intensity at 260 nm tocorrect for slight variations in the absolute amount of DNA present.Incorporation of labeling reagent is dependent on the amount of labelingreagent used, and is nearly linear before falling off at the higherratios.

EXAMPLE 7

[0207] The labeling method is highly sensitive, 0.1 pg of DNA can bedetected

[0208] (1) Materials

[0209] 1. FIG. 1(g)

[0210] 2. pDNA ( 3.3 μg/μL pCIluc; prepared according to Danko, I.,Williams, P., Herweijer, H. et al. Hum. Mol. Genetics (1997) in press.)

[0211] 3. Calf Thymus DNA (Sigma Chemical Co.)

[0212] 4. Anti-Digoxigenin-AP Fab fragments (Boebringer MannheimCorporation)

[0213] 5. CSPD chemiluminescence substrate (Boehringer MannheimCorporation)

[0214] 6. nylon filter (Micron Separations Inc, Westborough, Mass.)

[0215] (2) Procedure

[0216] pCIluc pDNA was labeled using FIG. 1(g) according to theprocedure given in example 1 using a 60 minute incubation period at 37°C., and a labeling reagent to DNA ratio of 5:1. The labeled DNA wasapplied to a nylon filter using a slot blot apparatus. Samples wereapplied to the nylon filter using 500 μL aliquots containing from 0.10pg to 50.0 pg labeled DNA. A 50 pg unlabeled control DNA (pCIluc andcalf thymus) samples were also applied to the nylon filter. FIG. 1(g)(50 pg) was treated according to example 1 in a tube without DNA, andapplied to the nylon filter as a purification control. The filter wasbaked at 70° C. for 1 hour and processed using anti-digoxigenin Fabfragments and the CSPD chemiluminescence substrate following proceduresdescribed in Boehringer Mannheim's “The DIG System User's Guide forFilter Hybridization,” chapter 9, pages 58-60. The filter was visualizedby exposing to X-ray film for 10 minutes.

[0217] (3) Results

[0218] The labeling method is highly sensitive, labeled DNA was visibleand above background from 0.1 pg to 50 pg.

EXAMPLE 8

[0219] Labeled DNA can hybridize to complementary DNA

[0220] DNA modified using FIG. 1(g) (the digoxin containing labelingreagent) was used to demonstrate hybridization of probe DNA tocomplimentary DNA immobilized on a nylon filter.

[0221] (1) Materials

[0222] 1. pDNA (3.3 μg/μL pCIluc; prepared according to Danko, I.,Williams, P., Herweijer, H. et al. Hum. Mol. Genetics (1997) in press.)

[0223] 2. Calf Thymus DNA (Sigma Chemical Co.)

[0224] 3. FIG. 1(g)

[0225] 4. Anti-Digoxigenin-AP Fab fragments (Boebringer MannheimCorporation)

[0226] 5. CSPD chemiluminescence substrate (Boehringer MannheimCorporation)

[0227] 6. nylon filter (Micron Separations Inc.)

[0228] (2) Procedure

[0229] pDNA (pCIluc) was labeled using FIG. 1(g) according to theprocedure given in example 1 using a labeling reagent to DNA ratio of1:1. The labeled DNA was alkali denatured and used to probe unlabeledpDNA (pCIluc) and calf thymus control DNA. The unlabeled DNAs weredenatured and fixed onto a nylon membrane at concentrations from 10 pgto 20 ng using a slot-blot apparatus. After blotting, the membrane wasbaked at 70° C. for 2 hr, the membrane was prehybridized and hybridizedaccording to the procedure given in Boehringer Mannheim's “The DIGSystem User's Guide for Filter Hybridization,” chapter 8, pages 45-48.The denatured, labeled probe DNA was diluted to a final concentration of20 ng/mL in the hybridization solution. The membranes were incubated inthe hybridization solution at 42° C. overnight. The membranes were thenprocessed following the procedure given in example 7.

[0230] (3) Results

[0231] The pDNA probed with complimentary labeled DNA was detectable at10 pg, the lowest level tested. No hybridization of the probe DNA to thenegative control (calf thymus DNA) was observed.

EXAMPLE 9

[0232] Labeled DNA can be used as probe DNA for southern hybridization

[0233] (1) Materials

[0234] 1. Mouse genomic DNA (generously provided by Hans Herweijer,University of Wisconsin-Madison)

[0235] 2. Human genomic DNA (Panvera Inc., Madison, Wis.)

[0236] 3. Calf Thymus DNA (Sigma Chemical Co.)

[0237] 4. Rat Beta Actin DNA (Panvera Inc.)

[0238] 5. Restriction enzyme Eco R1 (Promega Inc., Madison, Wis.)

[0239] 6. nylon filter (Micron Separations Inc.)

[0240] (2) Procedure

[0241] Hybridization of the labeled probe to target DNA was performedusing standard southern blot procedures as described in Maniatis'“Molecular Cloning,” chapter 9, pages 9.31 to 9.58. Briefly, probe DNAwas labeled with FIG. 1(g) in a 1:1 ratio according to example 1.Genomic DNAs were digested with Eco R1, run on an agarose gel, andtransferred to a nylon filter. The nylon filter was hybridized with thelabeled probe DNA at 42° C. overnight. The nylon membrane was thendeveloped according to example 7.

[0242] (3) Results

[0243] The developed film showed one hybridization band between mousegenomic DNA and the labeled rat beta actin probe. A weaker band was alsoobserved in the lane containing calf thymus genomic DNA indicating somecross reactivity between the labeled rat beta actin probe and calfthymus genomic DNA.

EXAMPLE 10

[0244] Labeled DNA is stable to long term storage conditions

[0245] (1) Materials

[0246] 1. pDNA (pCIluc) labeled with reagent 5 at a labeling reagent toDNA ratio of 0.2:1 and 0.04:1.

[0247] (2) Procedure and Results

[0248] Portions of labeled pDNA were stored in 100 mM HEPES buffer pH7.5 at −20° C., and at 4° C. Aliquots (0.4 μg) were analyzed by agarosegel electrophoresis at the time of preparation, after 24 hours, after 1week, and after 1 month at the stated storage conditions. Gels werephotographed before and after ethidium bromide staining at the statedtime periods. The amount of fluorescence, and the ratio of thesupercoiled to relaxed plasmid bands were examined. No degradation wasobserved for the labeled DNA stored at −20° C. at either labeling level.The labeled DNA stored at 4° C. was stable for 1 week at the 0.2:1ratio, and for at least 1 month at the 0.04:1 ratio.

EXAMPLE 11

[0249] Labeling reagents can be covalently attached to DNA for use inDNA localization following cellular delivery

[0250] (1) Materials

[0251] 1. pDNA (pCIluc) labeled with reagent 5 at two concentrations

[0252] 2. TransIT LT-1 transfection reagent (Mirus Corporation, Madison,Wis.)

[0253] 3. Cells (NIH 3T3 immortalized mouse fibroblast, ATTC, Rockville,Md.)

[0254] 4. fluorescence microscope (Leitz Orthoplan, Leitz Corporation,Germany)

[0255] (2) Procedure

[0256] Transfections were performed according to manufacturersrecommendations. 2 μg DNA were transfected per 35 mm well. Cells werefixed, and analyzed by fluorescence microscopy after 1 hour incubation.

[0257] (3) Results

[0258] Labeled DNA was observed in a punctate, perinuclear pattern.Strong fluorescent signal with low background was observed at bothconcentrations. No fluorescence was observed in cells transfected withunlabeled DNA.

[0259] The foregoing is considered as illustrative only of theprinciples of the invention. Further, since numerous modifications andchanges will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and operationshown and described. Therefore, all suitable modifications andequivalents fall within the scope of the invention.

1. A labeling molecule, for alkylating a chemical in a single-stepreaction thereby providing the chemical with a detectable reportermolecule, comprising: a covalently attachable labeling reagent having anet positive charge and a reactive group selected from the groupconsisting of mustard, aziridine, epoxide, episulfide, and cyclopropane.2. The labeling molecule of claim 1 wherein the mustard is selected fromthe group consisting, of nitrogen mustard and sulfur mustard.
 3. Thelabeling molecule of claim 1 wherein the reporter molecule comprises amolecule selected from the group consisting of fluorescence-emittingmolecules, hapten-containing molecules, proteins, and radioactivechemicals.
 4. The labeling molecule of claim 3 wherein the reportermolecule is linked to the reactive group via a spacer.
 5. The labelingmolecule of claim 4 wherein the spacer has a net charge greater thanzero.
 6. A kit comprising; a) a receptacle containing the labelingmolecule of claim 1; and, b) instructions for use.
 7. A compound havingthe structure comprising; D-B-A wherein, D is a label selected from thegroup consisting of fluorescence-emitting compounds, radioactivecompounds, haptens, immunogenic molecules, chemiluminescence-emittingcompounds, and proteins; B is a linker containing net charge greaterthan zero; and, A is an alkylating group selected from the groupconsisting of mustards, aziridines, epoxides, episulfides, andcyclopropanes.
 8. A compound having the structure comprising: D-B-Awherein, D is a label selected from the group consisting offluorescence-emitting compounds, radioactive compounds, haptens,immunogenic molecules, chemiluminescence-emitting compounds, andproteins; B is a linker containing a group with affinity for nucleicacid selected from the group consisting of DNA minor groove binder andDNA major groove binder; and, A is an alkylating group selected from thegroup consisting of mustards, aziridines, epoxides, episulfides, andcyclopropanes.